Baculovirus, including Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica nucleopolyhedrovirus (AcNPV), are a diverse family of arthropod viruses that are characterized by large (80 to 180 kb) circular double-stranded DNA (ds DNA) genomes and rodshaped enveloped virions. Both BmNPV and AcNPV are widely employed as vectors for the expression of foreign genes and pest control [1]. The advantage of the baculovirus-insect cell gene expression system in the protein production is that post-translational modifications are similar to those found in mammalian cells [2]. BmNPV has a 128,413 bp-long circular double-stranded DNA genome which encodes 136 potential genes [3]. The organization of the BmNPV genome closely ressembles that of AcNPV [4] which is the most extensively studied baculovirus. Mikhailov et al. [5] have purified and characterized a DNA-binding protein (designated DBP) from nuclear lysates of BmN cells (derived from Bombyx mori) infected with BmNPV. BmNPV DBP contains 317 amino acids and has an apparent molecular mass of 38 kDa. BmNPV DBP is encoded by the open reading frame 16 (ORF 16) located at nucleotides 16189 to 17139 in the BmNPV genome. This ORF is a homolog of AcNPV ORF 25 (96% homology), the product of which has not been identified so far. BmNPV DBP is found to bind preferentially to single-stranded DNA (ssDNA). The DNA binding site of DBP is estimated to be about 30 nucleotides per protein monomer. BmNPV DBP is capable of unwinding partial DNA duplexes in an in vitro system. Also, BmNPV DBP is found to be an early gene product during viral DNA replication (4–6 h postinfection) and it is one of the components of the multiprotein complex (with LEF-3 and IE-1) of the core replication machinery [6]. Thus, DBP appears to be crucial for viral DNA replication. Although baculoviruses, including BmNPV and AcNPV, efficiently replicate in the nuclei of arthropod cells, the dynamics and mechanism of viral DNA replication within the infected cell are currently poorly understood.
Mikhailov et al. [5] have purified the BmNPV DBP, and they, as well as Okano et al. [6] have studied the mechanism of viral DNA replication within the infected cell by using the rabbit polyclonal antibodies against an N-terminal six-His.Tag DBP. However, rabbit polyclonal antibodies are not adequate enough to recognize DBP specifically; monospecificity is needed to study the evolution of DBP and its role during viral DNA replication. Therefore, obtaining peptide ligands specific for DBP is necessary to address this issue, the reason for which the present research was initiated. Compared to the rabbit polyclonal antibodies against an N-terminal six-His.Tag DBP used by Mikhailov et al. [5] and Okano et al. [6], the peptide ligands specifically binding to DBP could be used as a valuable specific tool for diverse applications in basic as well as applied research.
The work reported herein provides the method for the amplification and cloning of the DBP gene from AcNPV, the construction of the expression plasmid for His.Tag AcNPV DBP, and its expression in BL21 (DE3) E. coli cells as well as the purification of the obtained recombinant His.Tag AcNPV DBP by using a column of Ni-NTA His.BindR Resin. The obtained purified His.Tag AcNPV DBP is then used as a target molecule for the selection of the peptide ligands specific for DBP from the FliTrx™ random peptide display library.
The FliTrx™ random peptide display library technique has the most desirable properties of available peptide selection technologies [7]. Understanding interactions between macromolecules is a central theme of biology—with these molecules, complementary in the surface character and shape, usually defining both the specificity and the strength of mutual interactions. Previously, the best method for precisely defining these contact surfaces was to determine the tertiary structure of an interacting complex by X-ray diffraction or by multi-dimensional NMR techniques. However, these approaches are time consuming and are not always feasible. Smith [15] and other researchers [16, 17] thus tried to pioneer a different method and succeeded in enabling huge population of diverse macromolecules to be screened, and specific members of these populations were selected on the basis of their binding affinity to an immobilized target. In this technique, DNA sequences encoding highly diverse libraries of short peptides are fused to the 5′-ends of bacteriophage coat protein genes. Following expression, these fusions were folded and assembled, exposing the random peptides on the bacteriophage surface. The phage/peptide libraries were then given the opportunity to bind to an immobilized target protein, typically a monoclonal antibody; and phage displaying peptides that interact specifically with the target were selectively retained through a washing procedure. Retained phage particles were eluted and then submitted for additional technique; its variations were applied to map a wide range of protein-protein interactions [18–22]. Although the peptide sequence information derived from these studies is useful, the ability to perform structural studies on the obtained peptides is limited both by the low expression levels of phage coat protein genes and by the character of the peptides selected by these systems, which are usually unconstrained molecules possessing many degrees of conformational freedom. To address both of these problems, Lu et al. [7] recently developed an alternative to Smith's display method [15], the FliTrx™ random display library technique. This approach entails the use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. The entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FliTrx) was efficiently exported and assembled into partially functional on the bacteria surface. A diverse library of random dodecapeptides was displayed in FliTrx on the exterior of E. coli as conformationally constrained insertion into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin.